The biosynthesis and secretion of penicillinase by Bacillus licheniformis and of the glycoprotein enzymes invertase and alkaline phosphatase by Saccharomyces yeasts, will be studied to determine the various steps in enzyme secretion by microorganisms and its relation to the process in animal cells. We will seqeunce and further characterize the in vitro precursor(s) of penicillinase obtained with an in vitro protein synthesizing system from Escherichia coli, and will than shift to a newly-developed system from B. licheniformis to follow the steps in processing the translation product to the final exoenzyme. Also we will investigate the relationship between this precursor and the phospholipoprotein penicillinase previously isolated from the cell membrane. The large exoenzyme (33,000 daltons) recently isolated will be characterized, especially in relation to the membrane enzyme. Because of the sensitivity of invertase to aromatic amines and to sodium borohydride, the possible presence of pyruvate residues in this protein will be investigated. Mutant D10-ER1, which produces primarily the large glycoprotein invertase (SUC1) and very little of the internal small form, is also of interest in that the large invertase is temperature-sensitive. Genetic studies will be carried out to determine the relation between the temperature-sensitivity of the enzyme and the selective deficiency of small enzyme with the overall purpose of determining if the mutation is in the structural gene for invertase. Studies on yeast alkaline phosphatase will focus on whether the active carbohydrate-free form produced in the presence of tunicamycin has a different location in the cell than the mature enzyme that is present largely in the vacuole.